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ORIGINAL RESEARCH ARTICLE
Journal of
OSU-03012 and Viagra Treatment
Inhibits the Activity of Multiple
Chaperone Proteins and Disrupts
the Blood–Brain Barrier: Implications
for Anti-Cancer Therapies
Cellular
Physiology
LAURENCE BOOTH,1 JANE L. ROBERTS,1 MEHRAD TAVALLAI,1 AIDA NOURBAKHSH,2
JOHN CHUCKALOVCAK,3 JORI CARTER,4 ANDREW POKLEPOVIC,4 AND PAUL DENT1*
1
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia
2
Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia
3
Bio-Rad Laboratories, Hercules, California
4
Department of Medicine, Virginia Commonwealth University, Richmond, Virginia
We examined the interaction between OSU-03012 (also called AR-12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role
of the chaperone glucose-regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than
additive fashion with OSU-03012 to kill stem-like GBM cells. Treatment of cells with OSU-03012/sildenafil: abolished the expression of
multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other
HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent
upon enhanced PERK-eIF2a-ATF4-CHOP signaling and was blocked by GRP78 over-expression. In vivo OSU-03012/sildenafil was more
efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced
expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU-03012/sildenafil synergized with low concentrations of
sorafenib to kill tumor cells, and with lapatinib to kill ERBB1 over-expressing tumor cells. In multiplex assays on plasma and human tumor
tissue from an OSU-03012/sildenafil treated mouse, we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as
response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling
profoundly enhanced OSU-03012/sildenafil lethality.
J. Cell. Physiol. 230: 1982–1998, 2015. ß 2015 Wiley Periodicals, Inc. This is an open access article under the terms of the Creative
Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work
is properly cited, the use is non-commercial and no modifications or adaptations are made.
OSU-03012, is a derivative of the drug celecoxib (Celebrex),
and lacks cyclooxygenase (COX2) inhibitory activity (Zhu
et al., 2004; Johnson et al., 2005). COX2 is over-expressed in
several tumor types and drugs that inhibit COX2, that is,
celecoxib have been shown to cause tumor cell-specific
increases in cell death, and that are also associated with a lower
rate of growth (Koehne and Dubois, 2004; Cui et al., 2005;
Kang et al., 2006; Klenke et al., 2006). Prolonged treatment
with COX2 inhibitors can reduce the incidence of developing
cancer, which, in addition, argues that COX2 inhibitors have
cancer preventative effects (Kashfi and Rigas, 2005; Narayanan
et al., 2006). Expression levels of COX2 do not simplistically
correlate with tumor cell sensitivity to COX2 inhibitors (Kulp
et al., 2004; Patel et al., 2005). Thus, COX2 inhibitors must
have additional cellular targets to explain their anti-tumor
biological actions.
Compared to the parent drug celecoxib, OSU-03012
(developed by Dr. Ching-Shih Chen at Ohio State University in
2004 and also known as AR-12, under licence from Ohio State
University to Arno Therapeutics, NJ) has a greater level of bioavailability in pre-clinical large animal models to the parent
compound and has an order of magnitude greater efficacy at
killing tumor cells (Yacoub et al., 2006; Park et al., 2008; Booth
et al., 2012a). Based on encouraging pre-clinical data OSU03012 underwent Phase I evaluation in patients with solid and
liquid tumors. Studies from the initial Phase I trial noted that
ß 2 0 1 5 W I L E Y P E R I O D I C A L S , I N C .
Abbreviations: PDGF, platelet-derived growth factor; EGF, epidermal
growth factor; CEL, celecoxib also called Celebrex; OSU, OSU03012 also called AR-12; SIL, sildenafil also called Viagra; VAR,
vardenafil also called Levitra; COX, cyclooxygenase; P, phospho-;
ca, constitutively active; WT, wild type; PERK, PKR like
endoplasmic reticulum kinase; HSP, heat shock protein; GRP,
glucose-regulated protein.
The authors have no conflicts of interest to report.
Contract grant sponsor: Massey Cancer Center.
Contract grant sponsor: PHS grants from the National Institutes of
Health;
Contract grant numbers: R01-CA141704, R01-CA150214, R01DK52825, R01-CA61774.
*Correspondence to: Paul Dent, Department of Biochemistry and
Molecular Biology, Virginia Commonwealth University, 401 College
Street, Massey Cancer Center, Box 980035, Richmond, VA 232980035. E-mail: pdent@vcu.edu
Manuscript Received: 24 February 2015
Manuscript Accepted: 26 February 2015
Accepted manuscript online in Wiley Online Library
(wileyonlinelibrary.com): 3 March 2015.
DOI: 10.1002/jcp.24977
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AR-12 AND VIAGRA
the “C max after single dose was dose-proportional but
high PK variability was observed, likely due to inadequate
disintegration and dissolution of the formulation in the
stomach” (ASCO 2013 meeting. http://meetinglibrary.asco.
org/content/115148-132) The C max of OSU-03012 in plasma
after 1 day at the MTD of 800 mg BID was 1 to 2 mM. After
28 days of treatment, the C max was 2 to 3 mM with the peak
C max in some patients being 8 mM. Thus, even considering
the problems associated with differential OSU-03012 drug
absorption in different patients, our use of OSU-03012 in prior
in vitro studies and in the present manuscript of 1.0 to 8.0 mM
of the drug is clinically relevant.
Initially, the tumoricidal effects of OSU-03012 in
transformed cells were argued to be via direct inhibition of the
enzyme PDK-1, within the PI3K pathway (Zhu et al., 2004).
And, in the low micro-molar range in cells, it has been shown
that OSU-03012 can lower AKT phosphorylation, presumably
by PDK-1 inhibition. In our previous studies, inhibition of either
ERK1/2 or phosphatidyl-inositol 3 kinase signaling enhanced
the toxicity of OSU-03012 (Yacoub et al., 2006; Park et al.,
2008; Booth et al., 2012a,b). However, our data has also
strongly argued that OSU-03012 toxicity, and in addition its
radiosensitizing effects, could not simplistically be attributed to
suppression of AKT signaling (Yacoub et al., 2006; Park et al.,
2008; Booth et al., 2012a,b). Specifically, our prior studies
have argued that OSU-03012 killed tumor cells through
mechanisms, which involved enhanced endoplasmic reticulum
(ER) stress signaling through activation of PKR-like
endoplasmic reticulum kinase (PERK), down-regulation/
reduced half-life of the ER and plasma membrane localized
HSP70 family chaperone GRP78/BiP/HSPA5, and a caspaseindependent, cathepsin-dependent autophagy-dependent form
of tumor cell death (Yacoub et al., 2006; Park et al., 2008; Booth
et al., 2012a,b). One of the hallmarks of any potentially useful
anti-cancer drug is that it is found to be relatively non-toxic
to “normal” cells/tissues and we previously noted that OSU03012, alone or in combination with other modalities had an
excellent therapeutic window comparing toxicity in normal
non-transformed cells to tumor cells both in vitro and in vivo.
ER stress signaling is mediated by three proximal sensors,
PERK, the IRE1 (inositol-requiring protein 1a)/X-box binding
protein 1 (XBP1) system and activating transcription factor 6
(ATF6). GRP78 plays a key role in regulating the ER stress
response; under resting conditions the majority of GRP78 is
associated with PERK and IRE1 and keeps these proteins in an
inactive state (Rao et al., 2012; Gorbatyuk and Gorbatyuk,
2013; Roller and Maddalo, 2013). GRP78, as a chaperone, also
plays an important role in the protein folding processes that
occur in the ER including during cancer, liver disease, and virus
replication (Roux, 1990; Earl et al., 1991; Anderson et al., 1992;
Hogue and Nayak, 1992; Carleton and Brown, 1997; Xu et al.,
1997, 1998; Bolt, 2001; Shen et al., 2002; Dimcheff et al., 2004;
He, 2006; Spurgers et al., 2010; Moreno and Tiffany-Castiglioni,
2014; Reid et al., 2014). When high levels of unfolded protein
are present in the ER, for example, in a tumor cell or in a
virally infected cell, GRP78 disassociates from PERK and IRE1
resulting in their activation, and GRP78 binds to the unfolded
proteins in the ER as a chaperone (Lee, 2007; Luo and Lee,
2013; Chen et al., 2014, and references therein). Activation of
PERK-eIF2a signaling acts to prevent the majority of cellular
proteins from being synthesized and IRE1 signaling enhances
the expression of additional GRP78 protein. Virus infection
can cause a profound ER stress response, which could by
cyto-toxic or prevent virus protein synthesis, thus some
viruses make targeting proteins, similar to mammalian
GADD34 and Nck1, that relocate protein phosphatase 1 with
eIF2a thereby preventing eIF2a phosphorylation and high
levels of toxic ER stress signaling (Rathore et al., 2013).
As GRP78 chaperones the unfolded protein(s), free GRP78
JOURNAL OF CELLULAR PHYSIOLOGY
eventually becomes available to re-associate with PERK and
IRE1 thereby shutting off the signaling system (Lee, 2007; Luo
and Lee, 2013; Chen et al., 2014). Of note, however, is that
prolonged ER stress signaling downstream of PERK and IRE1
can facilitate transformed cell killing, though in our laboratory
not primary cell killing (see above), arguing that a prolonged
reduction of GRP78 in transformed cells reduces cell viability
(Booth et al., 2014a,b).
We have published several manuscripts showing that knock
down/inhibition of PERK signaling suppressed OSU-03012
toxicity but that this effect appeared to be only partially eiF2a
dependent as the ability of OSU-03012 to cause high PERK
phosphorylation was not reflected in the more modest
phosphorylation increase of eIF2a. The induction of PERK
activity by OSU-03012 was associated with reduced GRP78
and HSP90 expression and increased HSP70 expression. More
recently, however, we have noted that the ability of sildenafil
to strongly enhance OSU-03012 or celecoxib lethality was
dependent on an even greater increase in PERK activity,
which was now associated with very high levels of eIF2a
phosphorylation.
Tumors of the brain are difficult to control. Untreated adult
glioblastoma (GBM) patients have a mean survival of several
months that is only prolonged up to 12–16 months by
aggressive therapeutic intervention. New therapeutic
approaches that could translate to the clinic for this malignancy
are urgently required. In two recently published studies, we
demonstrated that phosphodiesterase 5 (PDE5) inhibitors
enhanced the toxicity of standard of care chemotherapies in
bladder and pediatric CNS tumors (Booth et al., 2014c,d;
Roberts et al., 2014). In two additional even more recent
studies, we then noted that celecoxib or OSU-03012 lethality is
also enhanced by PDE5 inhibitors (Booth et al., 2014a,b). As
celecoxib, OSU-03012 and PDE5 inhibitors all readily cross the
blood–brain barrier, the present studies have attempted to
further understand how celecoxib and OSU-03012 lethality is
regulated by PDE5 inhibitors in malignant brain tumor cells,
with specific attention to the regulation of GRP78 expression
and activity of other HSP90 and HSP70 family chaperones.
Materials and Methods
Materials
Phospho-/total-antibodies were purchased from Cell Signaling
Technologies (Danvers, MA) and Santa Cruz Biotech. (Santa Cruz,
CA). All drugs, including OSU-03012, were purchased from
Selleckchem (Houston, TX). Commercially available validated
short hairpin RNA molecules to knock down RNA/protein levels
were from Qiagen (Valencia, CA). At least two different validated
siRNA molecules were independently used to confirm the effects
observed were not due to non-specific effects. Antibody reagents,
other kinase inhibitors, caspase inhibitors cell culture reagents, and
non-commercial recombinant adenoviruses have been previously
described (Booth et al., 2014a,b,c,d). The plasmid to express
GRP78/BiP/HSPA5 was kindly provided to the Dent Laboratory by
Dr. A.S. Lee (University of Southern California, Los Angeles, CA).
Previously characterized semi-established GBM5/GBM6/GBM12/
GBM14 GBM cells were supplied by Dr. C.D. James (University
of California, San Francisco) and Dr. J.N. Sarkaria (Mayo Clinic,
Rochester MN) and were not further characterized by ourselves
(Giannini et al., 2005).
Methods
Mammalian cell culture and in vitro exposure of cells
to drugs. All fully established cancer lines were cultured at 378C
(5% (v/v CO2) in vitro using RPMI supplemented with 10% (v/v)
fetal calf serum and 10% (v/v) non-essential amino acids. All
primary human GBM cells were cultured at 378C (5% (v/v) CO2)
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BOOTH ET AL.
in vitro using RPMI supplemented with 2% (v/v) fetal calf serum
and 10% (v/v) non-essential amino acids at 378C (5% (v/v CO2).
GBM5/6/12/14 stem cells were cultured in StemCell Technologies
NeuroCult NS-A Basal Medium supplemented with 20 mg/ml
bFGF, 20 mg/ml epidermal growth factor (EGF), and 2 mM heparin.
CD133þ glioma cells from this population were isolated by
fluorescence-activated cell sorting analysis. Cells, for example,
GBM12, grew as neurospheres and were characterized for
multiple stem cell markers, including CD44, SOX2, CD133, CD15,
CD36, Integrin B6, and MAP2. Neurosphere GBM cells had an
approximate 10-fold greater tumorigenicity in vivo than parental
wild type (WT) GBM cells (data not shown). For short-term cell
killing assays and immunoblotting, cells were plated at a density of
3 103 per cm2 and 24 h after plating were treated with various
drugs, as indicated. In vitro small molecule inhibitor treatments
were from a 100 mM stock solution of each drug and the maximal
concentration of vehicle (DMSO) in media was 0.02% (v/v). Cells
were not cultured in growth factor free media during any study.
Cell treatments, SDS–PAGE, and Western blot
analysis. Cells were treated with various drug concentrations,
as indicated in the figure legends. Samples were isolated at the
indicated times and SDS–PAGE and immunoblotting was
performed as described in Booth et al. (2014a,b,c,d). Immunoblots
were observed by using an Odyssey IR imaging system (LI-COR
Biosciences, Lincoln, NE).
Recombinant adenoviral and other virus vectors;
infection in vitro. We generated and purchased previously
noted recombinant adenoviruses as per Booth et al. (2014a,b,c,d).
Cells were infected with these adenoviruses at an
approximate m.o.i. as indicated in the figure/legend (usually an m.o.
i. of 0.1–50). Cells were incubated for 24 h to ensure adequate
expression of transduced gene products prior to drug exposures.
Viruses were purchased from Research BioLabs (Philadelphia, PA).
Detection of cell death by trypan blue assay. Cells
were harvested by trypsinization with Trypsin/EDTA for 10 min
at 378C. Harvested cells were combined with the culture media
containing unattached cells and the mixture centrifuged (800 rpm,
5 min). Cell pellets were resuspended in PBS and mixed with
trypan blue agent. Viability was determined microscopically using a
hemocytometer (a,b,c,d). Five hundred cells from randomly
chosen fields were counted and the number of dead cells was
counted and expressed as a percentage of the total number of cells
counted. Cell killing was confirmed using the Sceptor instrument
(Millipore, Billerica, MA), which measured tumor cell size/sub G1
DNA as an indication of tumor cell viability.
Cell death measurements by live/dead assay. Cells
were grown in 96-well plates with each well containing 10,000
cells in 200 ml of media. Cells were treated with the indicated
concentrations of drugs for the indicated amounts of time in each
panel. Plates were then centrifuged (500 rpm, 5 min) to re-adhere
floating dead cells to the base of each well. The media was removed
and live/dead assay reagent added and cells incubated for 10 min
before the reagent was removed. Cells were imaged in a Hermes
WiScan instrument under 10 magnification. Green cells ¼ viable;
yellow/red cells ¼ dieing/dead. The numbers of viable and dead
cells were counted manually from several images taken from one
well together with images from another two wells.
Plasmid transfection.
Plasmids. Cells were plated as described above and 24 h after
plating, transfected. Plasmids (0.5 mg) expressing a specific mRNA
or appropriate vector control plasmid DNA was diluted in 50 ml
serum-free and antibiotic-free medium (1 portion for each
sample). Concurrently, 2 ml Lipofectamine 2000 (Invitrogen), was
diluted into 50 ml of serum-free and antibiotic-free medium.
Diluted DNA was added to the diluted Lipofectamine 2000 for
each sample and incubated at room temperature for 30 min. This
mixture was added to each well/dish of cells containing 200 ml
serum-free and antibiotic-free medium for a total volume of 300 ml
and the cells were incubated for 4 h at 378C. An equal volume of
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2 medium was then added to each well. Cells were incubated for
48 h, then treated with drugs. To assess transfection efficiency of
plasmids, we used a plasmid to express GFP and defined the
percentage of cells being infected as the percentage of GFPþ cells.
For all cell lines, the infection efficiency was >70%.
siRNA. Cells were plated in 60 mm dishes from a fresh culture
growing in log phase as described above, and 24 h after plating
transfected. Prior to transfection, the medium was aspirated and
1 ml serum-free medium was added to each plate. For transfection,
10 nM of the annealed siRNA, the positive sense control doubled
stranded siRNA targeting GAPDH or the negative control (a
“scrambled” sequence with no significant homology to any known
gene sequences from mouse, rat, or human cell lines) were used
(predominantly Qiagen; occasional alternate siRNA molecules
were purchased from Ambion, Inc., Austin, TX). At least two
different validated siRNA molecules were independently used to
confirm the effects observed were not due to non-specific effects.
Ten nanomolar of siRNA (scrambled or experimental) was diluted
in serum-free media. Four microliters Hiperfect (Qiagen) was
added to this mixture and the solution was mixed by pipetting up
and down several times. This solution was incubated at room
temperature for 10 min, then added drop-wise to each dish. The
medium in each dish was swirled gently to mix, then incubated at
378C for 2 h. One milliliter of 10% (v/v) serum-containing medium
was added to each plate, and cells were incubated at 378C for
24–48 h before re-plating (50 103 cells each) onto 12-well plates.
Cells were allowed to attach overnight, then treated with drugs
(0–48 h). Trypan blue exclusion assays and SDS–PAGE/
immunoblotting analyses were then performed at the indicated
time points.
Animal studies
Athymic mice were treated by oral gavage with vehicle
(cremophore); celecoxib (50 mg/kg) þ sildenafil (10 mg/kg);
OSU-03012 (50 mg/kg) þ sildenafil (10 mg/kg) for 14 days QD
after which animals were humanely sacrificed and normal tissue/
organs obtained. Organs were fixed and 10 mm sections taken,
de-parafinized and H&E stained (special thanks to Dr. Hope
Richards, VCU Department of Pathology). Color images were
taken at 10 magnification (special thanks to Dr. Steven Grant,
VCU, Department of Hematology/Oncology). Athymic nude mice
(20 g) were injected with 1 107 BT474 cells into their fourth
mammary fat pad. Seven days after injection, animals with
50 mm3 tumors were treated by oral gavage with vehicle
(cremophore); celecoxib (10 mg/kg) þ sildenafil (5 mg/kg); OSU03012 (10 mg/kg) þ sildenafil (5 mg/kg) for 5 days after which
normal tissues were taken for IHC (GRP78, ABCB1, and ABCG2)
and the tumors and blood isolated for multiplex assays.
Data analysis
Comparison of the effects between various in vitro drug
treatments was performed after analysis of variance using the
Student’s t-test. Differences with a P-value of <0.05 were
considered statistically significant. Experiments shown are the
means of multiple individual points from multiple studies ( SEM).
Results
GBM stem cells compared to parental non-selected GBM cells
exhibited higher basal levels of phosphorylated eIF2a, but stem
cells nevertheless also expressed more GRP78/BiP/HSPA5
protein (Fig. 1A). Stem cell-like GBM cells expressed
considerably more MCL-1 and sphingosine-1-phosphate
receptor 1 (EDG-1) than parental WT cells. Of note, the
intensity of each image was normalized to the most intense
staining image for each antibody probe; WT cells did express
MCL-1, EDG-1, and GRP78 albeit at lower levels than stem
Fig. 1. OSU-03012 and sildenafil interact to kill tumor cells regardless of stem-like or anoikis-like properties, and that the tumor response
to this combination provides biomarkers for the addition of a third agent. A: GBM5/6/12/14 cells or stem-like selected variants of these cells
were plated in 96-well plates and 24 h after plating cells were cyto-centrifuged to sediment cells to the plate, and cells were fixed with 2%
paraformaldehyde containing Triton X100 for permeabilization. Immuno-fluorescence was performed to determine the expression of
GRP78/MCL-1/P-eIF2a, EDG-1, and counter-staining was with DAPI and detected using a Hermes Wiscan machine. The brightness/contrast
for each IF image was set to show the most intense staining and all other images were presented with the identical same brightness/contrast
setting. B: GBM cells or stem-like variants of these cells were treated with vehicle; celecoxib (5 mM) þ sildenafil (2 mM); or OSU-03012
(1 mM) þ sildenafil and the viability of cells determined by trypan blue exclusion assay after 24 h. (n ¼ 3, SEM) and P < 0.05 greater than
corresponding value in celecoxib treated cells; $P < 0.05 greater than corresponding value in wild type cells; @P < 0.05 less than corresponding
value in wild type cells. C: Athymic mice were treated with vehicle (Cremophore); OSU-03012 (50 mg/kg) þ sildenafil (10 mg/kg); celecoxib
(50 mg/kg þ sildenafil (10 mg/kg) for 14 days (nota bene: below in section (D) that anti-tumor effects were observed using half the doses of
these drugs and for half as long). Animals were sacrificed, the normal tissue organs collected and fixed. Sections (10 mm) were taken and H&E
stained by The Department of Pathology, VCU). D: Athymic nude mice were injected with 1 107 BT474 cells into their fourth mammary fat
pad. Seven days after injection, animals with 50 mm3 tumors were treated by oral gavage with vehicle (cremophore); celecoxib (10 mg/
kg) þ sildenafil (5 mg/kg); OSU-03012 (10 mg/kg) þ sildenafil (5 mg/kg) for 5 days after which tumors were isolated, and plated as single cells to
determine the ex vivo colony formation ability of in vivo treated tumors (n ¼ 3 SEM). #P < 0.05 less than vehicle control; ##P < 0.05 less than
[CEL þ SIL] value. E–J: Tumors treated for 5 days in section (D) were isolated as was mouse blood and plasma collected. Multiplex assays were
performed on tumor lysates and on clarified mouse plasma to detect changes in signal transduction parameters and on plasma cytokine levels
(n ¼ 4 tumors SEM). The treatments are presented in groups of three bars, with bars on the left being vehicle control; bars in the center
being [OSU þ SIL]; and bars on the right being [CEL þ SIL]. P < 0.05 greater than vehicle control value; #P < 0.05 less than vehicle control
value. K: BT474 cells were treated with vehicle control; OSU-03012 (1 mM) and and sildenafil (2 mM); the FGFR inhibitor BGJ398 (1 mM); the
JAK1/2 inhibitor ruxolitinib (1 mM) as indicated for 12 h. Cells viability was determined using a live/dead assay in a Hermes WiScan machine
where red/yellow cells ¼ dead; green cells ¼ alive (n ¼ 3 SEM). P < 0.05 greater than corresponding value in vehicle control treated cells.
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BOOTH ET AL.
Fig. 1.
(Continued)
cells (Fig. 1A, lower images). In GBM cells, it was noted that
stem cell selected cells were modestly but significantly more
sensitive to OSU þ sildenafil also called Viagra (SIL) treatment
than parental WT cells. In the majority of GBM cell lines,
treatment using 1 mM OSU-03012 caused a greater toxic
response when combined with sildenafil than did treatment
with the parental compound 5 mM celecoxib and sildenafil.
Regardless of whether breast cancer cells were parental
or generated to be anoikis resistant, the relative ability of
celecoxib also called Celebrex (CEL) þ SIL treatment to kill
parental or anoikis cells preferentially appeared to be more
unpredictable than in GBM cells and stochastic in nature (data
not shown). Prolonged high concentration dosing of mice with
[OSU-03012 þ sildenafil] or [celecoxib þ sildenafil] did not
cause obvious frank damage to normal tissues from an athymic
mouse (Fig. 1C). In contrast, treatment of animals with lower
doses of [OSU-03012 þ sildenafil] or [celecoxib þ sildenafil]
significantly reduced tumor volumes after 5 days of treatment
and reduced the long-term viability of in vivo treated cells, as
judged using ex vivo colony formation assays (Fig. 1D, data not
shown).
Based on these findings, we obtained normal brain and liver
and obtained tumor material and plasma from [OSU03012 þ sildenafil] and [celecoxib þ sildenafil] treated animals
carrying human BT474 mammary tumors that had been treated
for 5 days with these drug combinations. We discovered that
both drug combination treatments increased the levels of GCSF, and for [CEL þ SIL] bFGF and GM-CSF (Fig. 1E,F).
Treatment with [OSU þ SIL] strongly enhanced expression of
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the inhibitor PAI-1 and reduced expression of its cognate
cytokine uPA (Fig. 1G). Both drug combinations reduced the
phosphorylation of ERBB1 and of the platelet-derived growth
factor (PDGF)Ra/b, but not VEGFR2 (Fig. 1H). The reduced
activity of ERBB1 and PDGFRa/b correlated with reduced
AKT, p70 S6K, and mTOR activity in drug treated tumors
(Fig. 1I). The reduced activity of signal transduction pathways
correlated with reduced activity of the transcription factors
p53; NFkB; CREB; and c-Jun, but not Smad2 (Fig. 1J). Based on
our biomarker data, we then determined whether inhibition of
specific additional pathways could enhance [OSU þ SIL]
lethality. Based on data examining G-CSF, bFGF, and GM-CSF
levels we determined whether the clinically relevant FGFR pan
inhibitor BGJ398 or the FDA approved JAK1/2 inhibitor
ruxolitinib enhanced [OSU þ SIL] lethality. Inhibition of FGFR
signaling profoundly enhanced [OSU þ SIL] lethality (Fig. 1K).
Inhibition of JAK1/2 signaling more modestly, though
significantly, also enhanced [OSU þ SIL] lethality.
Expression of ERBB1, ERBB2, and of ERBB3 and ERBB4 in
BT474 breast cancer cells was reduced by OSU þ SIL
treatment (Fig. 2A). Treatment of GBM cells with CEL þ SIL
and with OSU þ SIL rapidly reduced total expression of ERBB1
(GBM12) and ERBB1 vIII (GBM6) within 6 h, whereas
expression of c-MET was reduced to a much lesser extent and
in a cell-dependent fashion (Fig. 2B). Drug combination
exposures rapidly reduced expression of S1PR1 (EDG1) and
S1PR5 (EDG5) as they did in a cell type dependent fashion for
the IL6R and the IL8R (Fig. 2C,D). Expression of the IGF1R and
of TNFaR1 were also reduced by the drug combination
AR-12 AND VIAGRA
Fig. 2. Treatment of tumor cells with [OSU-03012 þ sildenafil] or [celecoxib þ sildenafil] rapidly reduces the expression of multiple growth
factor receptors. A: BT474 mammary carcinoma cells known to strongly over-express ERBB2 and: B–F: GBM5/6/12/14 cells; in 96-well plates
were treated with vehicle; celecoxib (5 mM) þ sildenafil (2 mM); or OSU-03012 (1 mM) þ sildenafil and after 6 h treatment were fixed with 2%
(v/v) paraformaldehyde containing Triton X100 to permeabilized cells. Immuno-staining of the indicated growth factor receptors and plasma
membrane drug transporters was performed using standard techniques and IF images detected using a Hermes Wiscan machine.
G,H: Normal tissues from mice treated in Figure 1 section (D), brain and liver, were fixed, sectioned and immune-stained to determine the
total expression of GRP78, ABCB1, and ABCG2. Images were examined under 10 magnification using a Hermes WiScan instrument.
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(Fig. 2E). Drug combination treatment also reduced the total
expression of multiple plasma membrane associated drug efflux
pumps; pumps that are generally over-expressed in recurrent
patient tumors and are partly responsible for chemotherapy
resistance as well as for a functional blood–brain barrier
(Fig. 2F). Identical data were obtained in vivo examining normal
brain and liver tissues (Fig. 2G,H). Similar data showing knock
down of ABCB1 and ABCG2 expression were also obtained in
stem cell selected variants of GBM5/6/12/14 (data not shown).
In previous studies, we have shown that expression of the
death receptor CD95 (FAS) does not alter after particular
these drug exposures and in fact becomes plasma membrane
localized and clustered, observations indicative of CD95
activation (Booth et al., 2014a,b).
As treatment of cells with CEL þ SIL or with OSU þ SIL
weakly reduced expression of c-MET compared to other
growth factor receptors, we determined whether a clinically
relevant inhibitor of c-MET (crizotinib) could enhance the
lethality of CEL þ SIL or with OSU þ SIL. In a cell type
dependent fashion, that did not correlate with basal expression
or down-regulation of c-MET protein, crizotinib enhanced
CEL þ SIL or OSU þ SIL lethality (Fig. 3A). The lethality of
CEL þ SIL was potently enhanced in all GBM cells by the multikinase inhibitor sorafenib (Fig. 3B,C). Unlike [OSU þ SIL]
treatment in Fig. 2, the JAK1/2 inhibitor ruxolitinib did not
significantly enhance CEL þ SIL toxicity and the IGF1R inhibitor
OSI-906 only enhanced CEL þ SIL toxicity in GBM6 and
GBM14 cells. In GBM cells that expressed ERBB1 and ERBB1
vIII, the ERBB1/2 inhibitor lapatinib strongly enhanced
CEL þ SIL lethality. In a previous manuscript, we demonstrated
that the histone deacetylase inhibitor and blocker of
sphingosine-1-phosphate receptor signaling, FTY720
(multiple sclerosis drug: Gilenya), enhanced CEL þ SIL lethality.
The more potent HDAC inhibitors AR-42, vorinostat
enhanced CEL þ SIL lethality in GBM cells with AR-42 being
most potent on a molar basis (Fig. 3D). Similar cell killing
data were obtained in multiple GI tumor cell types (Fig. 3E).
The lethality of the OSU þ SIL drug combination was also
enhanced by HDAC inhibitors (Fig. 3F,G). The multiple
sclerosis drug FT720 as well as another multiple sclerosis drug
(mono-methyl fumarate [MMF]) enhanced OSU þ SIL lethality
(Fig. 3H).
HDAC inhibitors increase the acetylation of HSP90, which
reduces its chaperone function. The acetylation of heat shock
protein (HSP) 90 was increased and the expression of the
HSP90 client protein c-FLIP-s reduced by AR-42 treatment
(Fig. 4A, left blots; in agreement with Panner et al., 2007). This
collectively argues that the combination of OSU þ SIL þ AR-42
is inhibiting the functions of both GRP78 and HSP90, which may
explain the strong killing interaction between the drugs.
Furthermore, over-expression of HSP90 did not significantly
protect cells from OSU-03012 toxicity as a single agent, though
HSP90 over-expression did modestly reduce the enhancement
of cell death caused by IRE1 knock down; this effect was
abolished by AR-42 (Fig. 4A, right graph). Sildenafil and
OSU-03012 interacted to decrease total protein expression of
HSP90a/b in multiple GBM cell isolates (Fig. 4A, upper IF
images). Thus OSU-03012 inhibits HSP90 function as a single
agent and combined with sildenafil also considerably reduces
HSP90 protein levels. Based on our findings showing the
effect of sildenafil and OSU-03012 on HSP90 function, we
determined whether a clinically relevant HSP90 inhibitor
(SNX2112/SNX5422, 250 nM in vitro; patient C max @
100 mg/m2 is 3 mM) could enhance the lethality of OSU03012 and of [OSU þ SIL] in GBM cells. SNX2112 at its IC90
concentration for HSP90 inhibition enhanced the toxicity of
[OSU þ SIL] (Fig. 4B).
As GRP78 function appeared to be playing a central role
in our studies, we further explored the impact of altered ER
JOURNAL OF CELLULAR PHYSIOLOGY
stress signaling on the lethality of our CEL þ SIL and OSU þ SIL
drug combinations. Knock down of PERK expression or
expression of a dominant negative (dn) PERK protein
suppressed single agent OSU lethality (Fig. 4C). Overexpression of GRP78 protected cells from single agent OSU
killing (Fig. 4D, lower graphs). Treatment of cells with
OSU þ SIL enhanced the phosphorylation of PERK to a greater
extent than OSU alone, and profoundly enhanced eIF2a
phosphorylation, which correlated with significantly reduced
expression of GADD34, Nck1, and GRP78 (Fig. 4D, upper
blots). Of note, OSU-03012 as a single agent modestly
increased the expression of GADD34 that was associated
with enhanced PERK phosphorylation but not with enhanced
eIF2a phosphorylation. Thus it is probable that GADD34 and
Nck1, via the localized actions of protein phosphatase 1,
attenuates the ER stress signal from PERK by facilitating
dephosphorylation of eIF2a and that this inhibitory effect
on eIF2a is overcome by the addition of sildenafil which
reduces, below basal levels, the expression of GADD34 and
Nck1, and de facto thus also eIF2a-associated PP1 activity.
To test this hypothesis further, cells were treated with
OSU þ SIL for 12 h and total eIF2a protein
immunoprecipitated; in cells treated with OSU þ SIL, the
amount of PP1 and PP1 activity
co-precipitating with eIF2a was reduced (data not shown).
Knock down of IRE1 and XBP-1 enhanced OSU þ SIL
lethality whereas knock down of ATF6 had no effect (Fig. 4E).
Previously, we have shown that OSU-03012 and celecoxib
both modestly enhance expression of the HSP70 chaperone
protein (Park et al., 2008). OSU-03012 treatment modestly
enhanced HSP70 chaperone expression in a cell type
dependent fashion, an effect that was abolished by co-exposure
of cells with sildenafil resulting in a profound suppression of
HSP70 levels (Fig. 4F). Treatment of cells with OSU-03012 and
sildenafil or celecoxib and sildenafil reduced expression of the
ER-localized chaperone proteins GRP58 and GRP94 (Fig. 4G,
H). Treatment of cells with OSU-03012 and sildenafil reduced
expression of the ER-localized chaperone proteins HSP27,
HSP40, and HSP60 (Fig. 4I).
Treatment of GBM cells with OSU þ SIL resulted in a large
increase in eIF2a phosphorylation compared to individual drug
treatments (Fig. 5A,B). The reduction in EDG-1 expression
(total and cell surface) caused by OSU þ SIL was blocked by
expression of a dn eIF2a S51A protein (Fig. 5C–G). Treatment
of cells with OSU þ SIL increased expression of ATF4 in an
eIF2a-dependent fashion (Fig. 6A,B). The reduction in EDG-1
expression caused by OSU þ SIL treatment was prevented by
knock down of ATF4 or of CHOP (GADD153) (Fig. 6C–F).
The OSU þ SIL-induced increase in CHOP expression was
prevented by expression of dn eIF2a and the down-regulation
of EDG-1 levels was blocked by over-expression of GRP78
(Fig. 7A–D). Expression of dn eIF2a also prevented the druginduced reduction in MCL-1 and BCL-XL expression (Fig. 7E,
data not shown). Over-expression of GRP78 prevented the
drug-induced increase in eIF2a phosphorylation arguing that
the kinase that phosphorylates eIF2a is probably inhibited by
GRP78 (Fig. 7F). Over-expression of GRP78 also prevented the
drug combination-induced rapid declines in the expression of
EDG-1, ERBB1, IL6R, IGF1R, and the PDGFRa/b, as well as
drug efflux/blood–brain-barrier ABC transporters, suggesting
that the reduction in receptor expression and transporter
expression is also a result of reduced GRP78 levels and
enhanced ER stress signaling (Fig. 7G,H). Knock down of
CHOP expression suppressed CEL þ SIL and OSU þ SIL
lethality (Fig. 7I).
In prior studies, we have shown that OSU-03012 reduces
the expression of GRP78 through protein destabilization/
lower half-life without significantly altering GRP78 promoter
activity and that celecoxib increases GRP78 expression, with
Fig. 3. Third drug combinations with [OSU-03012 þ sildenafil] and with [celecoxib þ sildenafil] to further enhance tumor cell killing.
A: GBM5/6/12/14 cells in 96-well plates were treated with vehicle; celecoxib (5 mM) þ sildenafil (2 mM); or OSU-03012 (1 mM) þ sildenafil, with
or without the drug crizotinib (1 mM) and after 12 h of treatment were subjected to live/dead assays where the percentage cell death is noted
below each image (n ¼ 3 SEM). P < 0.05 greater than death value in two drug treated cells. B,C: GBM5/6/12/14 cells in 96-well plates were
treated with vehicle; celecoxib (5 mM) þ sildenafil (2 mM), with or without the drugs: sorafenib (0.5, 2.0 mM); ruxolitinib (0.5 mM); OSI-994
(1 mM); lapatinib (1 mM), as indicated. After 12 h of treatment cells were subjected to live/dead assays where the percentage cell death is noted
below each image (n ¼ 3 SEM). P < 0.05 greater than death value in two drug treated cells. D–F: GBM5/6/12/14 cells or [Huh7, HEPG2
liver], [HT29, HCT116 colon], [PANC1, Mia Paca2 pancreatic] cells in 96-well plates were treated with vehicle; celecoxib (5 mM) þ sildenafil
(2 mM) or OSU-03012 (1 mM) þ sildenafil with or without the drugs: AR-42 (250 nM); vorinostat (500 nM); valproate (0.1 mM), as indicated.
After 12 h of treatment, cells were subjected to live/dead assays where the percentage cell death is noted below each image (n ¼ 3 SEM).
P < 0.05 greater than death value in two drug treated cells. G,H: GBM cells were treated with vehicle; celecoxib (5 mM) þ sildenafil (2 mM) or
OSU-03012 (1 mM) þ sildenafil with or without FTY720 (50 nM) with or without MMF (5 mM). After 12 h of treatment, cells were subjected to
live/dead assays.
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Fig. 4.
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AR-12 AND VIAGRA
both drugs causing similar amounts of PERK phosphorylation.
This implies that both drugs, that are very similar in structure,
are probably binding to GRP78 with OSU-0312 destabilizing
the protein and celecoxib likely inhibiting the chaperone
function of the protein, which both collectively result in
increased PERK activity. Treatment of GBM cells with
celecoxib and sildenafil increased GRP78 expression, that is,
sildenafil did not alter the ability of celecoxib to increase
GRP78 levels (Fig. 8A). Nevertheless, this increase in
GRP78 expression was associated with increased eIF2a
phosphorylation. Treatment of GBM cells with OSU-03012
and sildenafil reduced expression of GRP78 and strongly
enhanced eIF2a phosphorylation. Treatment of GBM cells with
OSU-03012 rapidly reduced cell surface and total expression
of GRP78, an effect that was further enhanced by sildenafil
(Fig. 8B–E). In dose–response studies, we discovered that
OSU-03012 þ sildenafil treatment more rapidly and
completely reduced the expression of GRP78, HSP70, and
HSP90 than OSU-03012 treatment alone (Fig. 8F). Similar data
were obtained in primary mouse hepatocytes, and of note, as
judged by DAPI staining no cell killing was observed (Fig. 8G).
There are at least four kinases that phosphorylate eIF2a: PERK;
PKR; GCN2; HRI. The nitric oxide synthase inhibitor L-NAME
abolished the ability of OSU-03012 to reduce GRP78 levels
(Fig. 8H). However, the cGMP-dependent kinase inhibitor
reduced the ability of both OSU-03012 and [OSU-03012 þ
sildenafil] to lower GRP78 levels arguing that OSU-03012 may
mediate some of its biologic effects through modulation of
cGMP signaling. Knock down of PERK uniformly reduced the
ability of OSU þ SIL to increase eIF2a phosphorylation in all
lines tested (Fig. 8I). However, in a cell isolate dependent
fashion, we also found that PKR and HRI could contribute to the
drug-induced eIF2a phosphorylation (Fig. 8I,J).
Discussion
The present studies were initiated to determine the role of the
ER chaperone protein GRP78/BiP/HSPA5 in the cell death
response of tumor cells exposed to the drugs celecoxib or
OSU-03012 when combined with PDE5 inhibitors. We
discovered that sildenafil (and other PDE5 inhibitors,
unpublished results) enhanced the ability of OSU-03012 to
suppress GRP78 expression and blocked the ability of
celecoxib to increase GRP78 expression. This was
associated with increased expression of ATF4 and CHOP and
elevated cell killing; with decreased expression of multiple
growth factor receptors as well as plasma membrane drug
transporters, effects that were blocked by GRP78 overexpression. Killing was evident in GBM, breast, and GI
tumor cells, and in cells selected for stem-ness and for
anoikis-resistance.
In a prior study, we demonstrated that the HDAC
inhibitor and S1P signaling inhibitor FTY720 enhanced the
toxicity of CEL þ SIL in vitro and in vivo (Booth et al., 2014b).
The toxicity of CEL þ SIL and OSU þ SIL treatments were
enhanced to a greater extent than FTY720 by high affinity
HDAC inhibitors such as the clinically relevant drugs AR-42
and vorinostat. Vorinostat is an FDA approved HDAC
inhibitor for lymphoma and AR-42 will in 2015 be re-entering
the clinic combined with the multi-kinase inhibitor pazopanib
for a phase I trial in sarcoma and renal carcinoma at The
Medical College of Virginia Hospitals, Richmond, Virginia
(Poklepovic and Dent, unpublished observations). HDAC
inhibitors were also shown to cause inactivating acetylation of
the HSP90 chaperone, implying that the combination of
OSU þ SIL þ AR-42 will probably reduce the chaperone
functions of both GRP78, HSP70, HSC70, and HSP90, resulting
in a tumor cell predisposed to the stimulation of cell death
processes.
For GBM cells, CEL þ SIL treatment only modestly downregulated c-MET expression in GBM5 and GBM12 cells and in
two of our four GBM isoaltes, GBM12 and GBM14, the c-MET
inhibitor crizotinib enhanced CEL þ SIL and OSU þ SIL
lethality. However, c-MET expression was effectively downregulated in GBM14 and not down-regulated in GBM5 arguing
that simplistic changes in receptor expression are not a valid
Fig. 4. The regulation of chaperone function and expression by OSU-03012, sildenafil and HDAC inhibitors. A: (Left blots) GBM6 cells were
treated with AR-42 (250 nM) for 90 min, after which HSP90 was immunoprecipitated and probed for total HSP90 expression and HSP90
acetylation. In parallel tumor, cell lysate was immunoblotted for the total expression of GAPDH and of c-FLIP-s. Right graph: GBM6 and
GBM12 cells were infected with an empty vector virus or a virus to express HSP90, and in parallel transfected with either a scrambled siRNA
(siSCR) or an siRNA to knock down IRE1 expression. Twenty-four hours after infection, cells were treated with vehicle or OSU-03012 (1 mM).
Cells were isolated after 24 h and viability determined by trypan blue exclusion assay (n ¼ 3 SEM). P < 0.05 greater than siSCR þ CMV value;
P < 0.05 greater than siIRE1 þ CMV value; #P < 0.05 less than siIRE1 þ CMV value; ?P > 0.90 between siSCR þ CMV and siSCR þ HSP90
values. IF images above graph: GBM6/12 cells were treated with vehicle; OSU-03012 (1 mM); sildenafil (2 mM), or the drugs in combination and
after 6 h treatment were fixed with 2% (v/v) paraformaldehyde containing Triton X100 to permeabilized cells. Immuno-staining of HSP90a/b
was performed using standard techniques and IF images detected using a Hermes Wiscan machine. B: GBM cells were treated for 12 h with
vehicle, [OSU-03012 (1 mM) þ sildenafil (2 mM)], SNX2112 (250 nM), or the three drugs combined. Cell viability under each condition was
determined by live/dead assays (n ¼ 3 SEM). C: GBM6 cells were transfected with siSCR or siRNA molecules to knock down expression of
IRE1, XBP1, and PERK. In parallel sets of plates, cells were transfected with empty vector plasmid (CMV) or to express dominant negative
PERK. Twenty-four hours after transfection, cells were treated with vehicle or with OSU-03012 (2 mM). Twenty-four hours after drug
treatment, cell viability was determined by trypan blue exclusion assay (n ¼ 3 SEM). #P < 0.05 lower than corresponding value in siSCR cells;
P < 0.05 greater than corresponding value in siSCR cells. D: (Upper blots) GBM6 cells were treated with OSU-03012 (1 mM), sildenafil (2 mM),
or the drugs in combination for 6 h after which cells were lysed. Immunoblotting and phospho-immunoblotting was performed to detect the
expression/phosphorylation of the indicated proteins. Lower Graphs: GBM6 and GBM12 cells were transfected with empty vector (CMV) or
to express GRP78. Twenty-four hours after transfection, cells were treated with 2 mM OSU-03012 and 24 h later viability determined via
trypan blue exclusion assay (n ¼ 3 SEM). #P < 0.05 less than corresponding value in CMV transfected cells. E: GBM6 cells were transfected
with scrambled control (siSCR) or siRNA molecules to knock down IRE1, XBP1, or PERK. Twenty-four hours after transfection, cells were
treated with OSU-03012 (1 mM), sildenafil (2 mM), or the drugs in combination for 24 h. Cell viability was determined by trypan blue exclusion
assay (n ¼ 3 SEM). P < 0.05 greater than OSU-03012 single agent treatment in siSCR cells; P < 0.05 greater than corresponding values in
siSCR cells; #P < 0.05 less than corresponding values in siSCR cells. F–I: GBM cells were treated with vehicle; celecoxib (5 mM), sildenafil
(2 mM), OSU-03012 (1 mM) as single agents or in the indicated combinations for 6 h. Cells were then fixed with 2% (v/v) paraformaldehyde
containing Triton X100 to permeabilize cells. Immuno-staining of total HSP70 and HSC70 expression combined (part F) or expression of
GRP94 and GRP58 (parts G and H) or expression of HSP27/HSP40/HSP60 (part I) was performed using standard techniques and IF images
detected using a Hermes Wiscan machine.
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Fig. 5.
JOURNAL OF CELLULAR PHYSIOLOGY
AR-12 AND VIAGRA
biomarkers, and that the phosphorylation level of c-MET may
be a much better predictor of whether a tumor cell has any
survival addition to signals from this receptor. As the diabetes
drug metformin has also been shown to reduce GRP78
expression, we believe this agent could also interact with
OSU-03012 and sildenafil to profoundly enhance OSU þ SIL
lethality.
Our prior studies have linked OSU-03012-induced ER stress
signaling by PERK as being causal in the toxicity of OSU-03012.
The present studies demonstrated that the drug combination
of CEL þ SIL or OSU þ SIL strongly activated both PERK and
eIF2a that was associated with decreased GADD34 and Nck1
and GRP78 expression. Reduced GADD34/Nck1 expression
will facilitate eIF2a signaling by reducing co-localization of
the Ser/Thr phosphatase PP1 with eIF2a. Knock down
of CHOP prevented drug combination down-regulation of
growth factor receptors and suppressed drug combination
lethality, demonstrating that enhanced ER stress signaling distal
to PERK and eIF2a was essential for drug combination toxicity.
As published previously, we noted that the IRE1 arm of the ER
stress response was protective against OSU-03012 and
OSU þ SIL treatment, and that the ATF6 arm appeared not to
significantly alter the response. Knock down of IRE1/XBP-1
also enhanced OSU-03012 and sildenafil combination toxicity.
Further studies will need to understand how and why IRE1
signaling in our system is protective, for example, modulation
of RIDD or XBP-1 function (Tong et al., 2011; Donnelly
et al., 2013; Sano et al., 2013; Maurel et al., 2014). Preliminary
studies suggest the drug combination regulates viability
downstream of IRE1 via altered EDEM expression (unpublished
observations).
We have previously shown that OSU-03012 as a single agent
increases the levels of autophagosomes and autolysosomes and
that this effect is significantly enhanced by sildenafil; enhanced
autophagy in this system is cytotoxic as judged by the ability
of ATG5 or Beclin1 knock down to prevent formation of
autophagic vesicles and to prevent tumor cell killing. We also
demonstrated that this elevated level of autophagy was
suppressed by over-expression of GRP78, and that GRP78
over-expression protected tumor cells. In the present
studies, we noted that the drug combination reduced the
expression of both MCL-1 and BCL-XL, and as we have
previously shown that this down-regulation can lead to
increased levels of unbound Beclin1, which stimulate
autophagy, the ER stress signaling pathway will likely lead to
increased toxic tumor cell autophagy as well as to increased
mitochondrial instability. Thus cell death pathways through
autophagy and caspase activation will become engaged
following OSU þ SIL treatment, suggestive that tumor cell
resistance mechanisms will be subverted and become
ineffective (Fig. 9).
Sildenafil was developed as an inhibitor of PDE5 with cardioprotective effects, and serendipitously became an approved
thereapeutic for erectile dysfunction (Zusman et al., 1999).
PDE5 expression is not confined to the corpus cavernosum
in the human penis and is expressed in the wider vasculature,
myocardium, and tumor cells (Karami-Tehrani et al., 2012;
Shan et al., 2012). We have independently validated that PDE5
is over-expressed in liver, colorectal, and lung cancer cells
compared to normal tissues, and that viral infection increases
PDE5 expression (unpublished observations). PDE5 catalyzes
the degradation of cyclic GMP; that is, thus PDE5 inhibitors
increase cGMP levels (Zhang et al., 2012). Nitric oxide (NO)
induces smooth muscle relaxation via the actions of cGMP
(Kato et al., 2012). NO at nanomolar levels binds tightly to a
heme group in NO-guanylyl cyclase (GC), also known as
soluble guanylyl cyclase, and causes a 150-fold activation of
the enzyme (Potter, 2011). Activation of NO-GC elevates
cGMP levels, which initiate the cGMP signaling pathway, in part
through activation of cGMP-dependent protein kinase (PKG)
(Friebe and Koesling, 2009). It is known in non-tumor cells that
cGMP/PKG, through its stimulatory actions upon the ERK, p38
MAPK, JNK, and NFkB pathways can increase the expression
of inducible nitric oxide synthase (iNOS) (Komalavilas et al.,
1999; Choi et al., 2007; Das et al., 2008). Thus, it is possible in
our system that increased levels of NO activate GC and
increase cGMP levels, that activates signaling pathways, which
increase iNOS levels; and increased iNOS levels lead to further
increases in cellular NO.
Elevation of cGMP or overexpression of constitutively active
PKG can result in phosphorylation and activation of the JNK
pathway and promote apoptosis (Thompson et al., 2000;
Deguchi et al., 2004; Zhu et al., 2005a, 2009; Zhu and Strada,
2007). Our prior findings showed that OSU þ SIL interacted to
cause toxic JNK activation (Booth et al., 2012b). High
concentrations of sildenafil and vardenafil induce caspasedependent apoptosis of B-chronic lymphocytic leukemia cells
but not in normal B cells, suggesting a tumor selective toxicity
of PDE5 inhibitors (Sarfati et al., 2003). PDE5 inhibitors
enhance tumor/vasculature permeability and efficacy of
chemotherapy in a rat brain tumor model (Black et al., 2008).
When transiently expressed in HT29 colon cancer cells,
constitutively activated mutants of PKG beta, inhibit colony
formation and induce apoptosis (Zhu et al., 2005b). In PC12
cells cGMP signaling, via activation of the AKT pathway
prevents apoptosis (Ha et al., 2003). Others have argued that
cGMP and NO kills cells through activation of the CD95/FAS-L
pathway (Hayden et al., 2001). These latter findings are similar
to the data in our most recent manuscript examining this drug
where OSU-03012 and sildenafil interacted to kill through
CD95 activation.
In conclusion, treatment of cells with OSU þ SIL rapidly
reduces the expression of the key chaperone protein GRP78,
as well as reducing the chaperone functions of multiple other
survival factors. The drug combination also largely abolishes
the blood–brain barrier.
Fig. 5. Celecoxib and sildenafil interact to increase eIF2a serine 51 phosphorylation which facilitates reduced expression of S1PR1 in GBM
cells. A,B: GBM6/12 cells were plated in 96-well plates and transfected with either empty vector or with a plasmid to express eIF2a S51A.
Twenty-four hours after transfection, cells were treated with vehicle, OSU-03012 (1 mM), sildenafil (2 mM), or the drugs in combination
for 6 h. Cells were fixed and permeabilized and immuno-fluorescence performed to determine the levels of phospho-S51 eIF2a. C–G: GBM6/
12 cells were plated in 96-well plates and transfected with either empty vector or with a plasmid to express eIF2a S51A. Twenty-four
hours after transfection, cells were treated with vehicle, celecoxib (5 mM)/OSU-03012 (1 mM), sildenafil (2 mM), or the drugs in combination,
or with FTY720 (50 nM), for 2, 4, and 6 h, as indicated. Cells were fixed and permeabilized or not permeabilized as indicated and
immuno-fluorescence performed to determine the levels of S1PR1 (EDG-1) in the plasma membrane and in the whole permeabilized cell
(n ¼ 3 SEM). #P < 0.05 less than vehicle treated value at that time point; $P < 0.05 less than corresponding value in CMV transfected cells.
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Fig. 6. Increased expression of ATF4 after [OSU-03012 þ sildenafil] treatment is eIF2a dependent and the drug-induced suppression of
EDG-1 expression requires ATF4/CHOP signaling. A,B: GBM cells were transfected with empty vector CMV or to express dominant negative
eIF2a. Twenty-four hours after transfection, cells were treated with vehicle, OSU-03012 (1 mM), sildenafil (2 mM), or the drugs in combination
for 6 h. Cells were fixed and permeabilized and immuno-fluorescence performed to determine the levels of ATF4. C–F: GBM cells were
transfected with a scrambled siRNA (siSCR) or siRNA molecules to knock down the expression of ATF4 or CHOP. Twenty-four hours after
transfection, cells were treated with vehicle, OSU-03012 (1 mM), sildenafil (2 mM), or the drugs in combination for 6 h. Cells were fixed and
permeabilized and immuno-fluorescence performed to determine the total cellular levels of S1PR1 (EDG-1).
JOURNAL OF CELLULAR PHYSIOLOGY
Fig. 7. Activation of eIF2a signaling promotes expression of CHOP and down-regulation of EDG-1 that is blocked by over-expression of
GRP78. A–D: GBM cells as indicated were transfected with empty vector (CMV), with a plasmid to express GRP78, or with a plasmid to
express dominant negative eIF2a. Twenty-four hours after transfection, cells were treated with vehicle, OSU-03012 (1 mM), sildenafil (2 mM),
or the drugs in combination for 6 h. Cells were fixed and permeabilized and immuno-fluorescence performed to determine the total cellular
levels of S1PR1 (EDG-1), and CHOP (GADD153). E: Tumor cells were transfected with empty vector CMV or to express dominant negative
eIF2a. Twenty-four hours after transfection, cells were treated with vehicle, OSU-03012 (1 mM), sildenafil (2 mM), or the drugs in combination
for 6 h. Cells were fixed and permeabilized and immuno-fluorescence performed to determine the levels of MCL-1. F: Tumor cells were
transfected with empty vector CMV or to express GRP78. Twenty-four hours after transfection, cells were treated with vehicle, OSU-03012
(1 mM), sildenafil (2 mM), or the drugs in combination for 6 h. Cells were fixed and permeabilized and immuno-fluorescence performed to
determine the levels of eIF2a serine 51 phosphorylation. G,H: Tumor cells were transfected with empty vector CMV or to express GRP78.
Twenty-four hours after transfection, cells were treated with vehicle or with OSU-03012 (1 mM) and sildenafil (2 mM) for 6 h. Cells were fixed
and permeabilized and immuno-fluorescence performed to determine the total cellular levels of the indicated growth factor receptors
and drug efflux pump proteins. I: GBM cells were transfected with a scrambled siRNA (siSCR) or an siRNA molecule to knock down the
expression of CHOP. Twenty-four hours after transfection, cells were treated with vehicle or with OSU-03012 (1 mM) and sildenafil (2 mM).
After 24 h of treatment, cells were subjected to live/dead assays where green cells are alive and yellow/red cells are dead.
JOURNAL OF CELLULAR PHYSIOLOGY
Fig. 8. Sildenafil facilitates a rapid further reduction in GRP78 protein expression levels in cells treated with OSU-03012. A: GBM cells were
treated with vehicle, celecoxib (5 mM)/OSU-03012 (1 mM), sildenafil (2 mM) or the drugs in combination for 6 h. Cells were fixed and
permeabilized and immuno-fluorescence performed to determine the total cellular levels of GRP78. B–E: GBM cells were treated with
vehicle, OSU-03012 (1 mM), sildenafil (2 mM), or the drugs in combination for the indicated amounts of time. At each time point, cells were
fixed with paraformaldehyde and portions of fixed cells also being permeabilized with Triton X100. F: HuH7 (hepatoma) and HT1080
(sarcoma) were treated with vehicle, OSU-03012 (0–3 mM), sildenafil (2 mM), or the drugs in combination. Cells after 2 and 6 h drug exposure
were fixed in place and permeabilized. Immuno-fluorescence was performed to determine the total cellular levels of GRP78; HSP90; HSP70.
G: Primary mouse hepatocytes (wild type C57 black or CHOP / C57 black) were plated and 12 h after plating treated with vehicle, OSU-03012
(1 mM), sildenafil (2 mM), or the drugs in combination. Six hours after drug exposure cells were fixed in place and permeabilized.
Immuno-fluorescence was performed to determine the total cellular levels of GRP78. H,I: GBM cells were transfected with scrambled
siRNA or siRNA molecules to knock down the expression of PERK, PKR, GCN2, or HRI. Twenty-four hours after transfection, cells were
treated with vehicle or OSU-03012 (1 mM) þ sildenafil and the cells fixed and permeabilized 6 h later. Immuno-fluorescence antibody staining
was performed to determine the total levels of phospho-eIF2a serine 51 under each condition.
JOURNAL OF CELLULAR PHYSIOLOGY
AR-12 AND VIAGRA
Fig. 9. Possible molecular mechanisms by which OSU-03012 (AR-12) and PDE inhibitors could kill tumor cells. The ability of OSU-03012
(AR-12) alone, or enhanced by a PDE5 inhibitor, to reduce GRP78 expression and the function of multiple other chaperones will prevent the
correct folding of essential tumor cell proteins resulting in dead tumor cells. The combination of OSU-03012 and a PDE5 inhibitor will also
reduce the expression of plasma membrane signaling receptors whose expression is essential for tumor cell growth and invasion. The loss of
GRP78 and other chaperones in tumor cells will result in much lower expression levels of key oncogenic kinases, including PI3K-AKT signaling
(Liu et al., 2013, 2015; Tsai et al., 2015). Collectively our data strongly argue that it is a strong possibility that our drug combination will abolish
the blood–brain barrier and have profound anti-tumor capabilities.
Acknowledgments
Support for the present study was funded in part by
philanthropic monies supplied by Massey Cancer Center (to
P.D.). Support for the present study was funded from PHS
grants from the National Institutes of Health [R01-CA141704,
R01-CA150214, R01-DK52825, and R01-CA61774]. Thanks
to Mrs. Grizzard for her support to the Dent lab and to Dr. H.
F. Young and the Betts family fund for support in the purchase
of the Hermes Wiscan instrument. P.D. is the holder of the
Universal, Inc. Chair in Signal Transduction Research. P.D.
wishes to thank Dr. Hylemon (VCU) for supplying primary
mouse hepatocytes.
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